PROJECT SUMMARY/ABSTRACT Human pluripotent stem cells (hPSCs) have enormous promise for regenerative medicine as they can differentiate into cells from all three embryonic germ layers. Further hPSCs provide a unique pre-clinical screening tool for evaluating disease mechanisms and therapies in patient samples. Despite the enormous potential of hPSC, many obstacles need to be overcome before the use of stem cells in cell-based therapy will be realized, including an incomplete knowledge of the growth factor cues regulating human skeletal muscle progenitor cell (SMPC) specification, growth and engraftment. In this proposal we will investigate the cellular cues required to direct skeletal muscle fate from hPSCs as seen during embryonic development. We will also develop the first screening approach to identify new regulators of human SMPC fate. It is imperative that we understand how to obtain SMPCs without genetic or viral manipulation. As an alternative approach we have developed a unique non viral reprogramming platform, which results in superior delivery and reprogramming efficiency, and will be utilized to specify SMPCs without viral or genetic manipulation. We will compare the in vivo engraftment potential of growth factor directed versus reprogrammed SMPCs in a mouse model of DMD. The development of reprogramming strategies to direct SMPC fate and the identification of signals regulating muscle progenitor cell specification and maintenance could improve our basic understanding of human skeletal myogenesis as well as enhance our ability to generate scalable progenitors from humans for muscle disorders including DMD.